PROJECT SUMMARY/ABSTRACT Down syndrome (DS) is one of the strongest risk factors for acute lymphoblastic leukemia (ALL), conferring a 20-fold increased risk compared to children without DS. Survival for children with DS-ALL remains 10-20% lower than that of non-DS-ALL patients, due to both relapse and toxicity. Children with DS also have an increased risk of several birth defects and chronic health conditions. Although these increased risks have been known for decades, the basis for the increased risk of leukemia remains unclear, and there is a particular paucity of studies exploring the interplay between other DS phenotypic features and ALL susceptibility. While increased dosage of chromosome 21 genes is likely contributory to ALL risk, trisomy 21 alone is not sufficient, as the lifetime risk of ALL in children with DS is under 2%. We hypothesize that additional genetic modifiers and other DS-related phenotypes in combination with trisomy 21 influence susceptibility to ALL. We have made several important observations to date. 1) In the first genome-wide association study of DS-ALL, we observed that loci in ALL susceptibility genes (e.g., CDKN2A, IKZF1) have stronger effects on ALL risk in children with DS compared to those without. 2) We have preliminary evidence that children with DS-ALL have a greater burden of structural birth defects than children with DS without ALL, perhaps suggesting an ALL- predisposition phenotype with additional syndromic features. These observations suggest unique patterns of ALL susceptibility in the background of trisomy 21. However, we have not yet systematically evaluated: 1) the role of structural, rare, and chromosome 21 variants, 2) the association between inherited genetic variation and somatic genomic abnormalities, or 3) the role of other DS-related phenotypes on leukemia susceptibility and outcomes. Through the NIH INCLUDE and Kids First mechanisms, we and collaborators have initiated large- scale genomic sequencing of 2,500 children with DS (~400 with ALL). Capitalizing on this unprecedented genomic profiling and unique DS cohort, the objectives of the current study are to determine the molecular underpinnings of ALL in children with DS; and to determine whether DS-related phenotypes are associated with risk of ALL and/or with outcomes (toxicities, relapse, and survival). To achieve these objectives, the aims of this study are to 1) perform a comprehensive analysis of heritable variation associated with risk of ALL in children with DS, with a focus on structural, rare, and chromosome 21 variants; and 2) conduct deep phenotyping of children with DS-ALL to identify the impact of DS-related phenotypes on leukemia susceptibility and outcomes. This project will address fundamental questions of why children with DS have an increased risk of ALL, how their leukemia differs from that of children without DS, and how other DS-related phenotypes may influence ALL susceptibility, survival, and toxicities. Findings from this study may lead to improved genetic testing and counseling strategies for children with DS. Insights into genes driving DS-ALL may guide development of targeted therapies.